Mass spectrometric determination of cookson-derivatized, non-metabolized vitamin d

ABSTRACT

The invention relates to the detection of vitamin D. In a particular aspect, the invention relates to methods for detecting derivatized vitamin D by mass spectrometry.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.Nos. 61/285,938 (filed Dec. 11, 2009) and 61/285,943 (filed Dec. 11,2009), both of which are incorporated herein by reference in theirentirety including all figures and tables.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of non-metabolizedvitamin D. In a particular aspect, the invention relates to methods forquantitative measurement of non-metabolized vitamin D by tandem massspectrometry.

BACKGROUND OF THE INVENTION

Vitamin D is an essential nutrient with important physiological roles inthe positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can bemade de novo in the skin by exposure to sunlight or it can be absorbedfrom the diet. There are two forms of vitamin D; vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is theform synthesized de novo by animals. It is also a common supplementadded to milk products and certain food products produced in the UnitedStates. Both dietary and intrinsically synthesized vitamin D₃ mustundergo metabolic activation to generate the bioactive metabolites. Inhumans, the initial step of vitamin D₃ activation occurs primarily inthe liver and involves hydroxylation to form the intermediate metabolite25-hydroxycholecalciferol (calcifediol; 25OHD₃). Calcifediol is themajor form of Vitamin D₃ in circulation. Circulating 25OHD₃ is thenconverted by the kidney to form 1,25-dihydroxyvitamin D₃ (calcitriol;1,25(OH)₂D₃), which is generally believed to be the metabolite ofVitamin D₃ with the highest biological activity.

Vitamin D₂ is derived from fungal and plant sources. Manyover-the-counter dietary supplements contain ergocalciferol (vitamin D₂)rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potencyprescription form of vitamin D available in the United States, isformulated with ergocalciferol. Vitamin D₂ undergoes a similar pathwayof metabolic activation in humans as Vitamin D₃, forming the metabolites25OHD₂ and 1,25(OH)₂D₂. Vitamin D₂ and vitamin D₃ have long been assumedto be biologically equivalent in humans, however recent reports suggestthat there may be differences in the bioactivity and bioavailability ofthese two forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol.Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare inclinical settings. Rather, serum levels of 25-hydroxyvitamin D₃,25-hydroxyvitamin D₂, and total 25-hydroxyvitamin D (“25OHD”) are usefulindices of vitamin D nutritional status and the efficacy of certainvitamin D analogs. The measurement of 25OHD is commonly used in thediagnosis and management of disorders of calcium metabolism. In thisrespect, low levels of 25OHD are indicative of vitamin D deficiencyassociated with diseases such as hypocalcemia, hypophosphatemia,secondary hyperparathyroidism, elevated alkaline phosphatase,osteomalacia in adults and rickets in children. In patients suspected ofvitamin D intoxication, elevated levels of 25OHD distinguishes thisdisorder from other disorders that cause hypercalcemia.

Measurement of 1,25(OH)₂D is also used in clinical settings. Certaindisease states can be reflected by circulating levels of 1,25(OH)₂D, forexample kidney disease and kidney failure often result in low levels of1,25(OH)₂D. Elevated levels of 1,25(OH)₂D may be indicative of excessparathyroid hormone or can be indicative of certain diseases such assarcoidosis or certain types of lymphomas.

Detection of vitamin D metabolites has been accomplished byradioimmunoassay with antibodies co-specific for 25OHD₂ and 25OHD₃.Because the current immunologically-based assays do not separatelyresolve 25OHD₂ and 25OHD₃, the source of any nutritional deficiency ofvitamin D cannot be determined without resorting to other tests. Reportshave been published that disclose methods for detecting specific vitaminD metabolites using mass spectrometry. In some of the reports, thevitamin D metabolites are derivatized prior to mass spectrometry, but inothers, they are not. For example Holmquist, et al., U.S. patentapplication Ser. No. 11/946,765, filed Dec. 28, 2007; Yeung B, et al., JChromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids. 2000,65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001, 24(7):738-43;Higashi T, et al., J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T,et al., Anal. Biochanal Chem, 2008, 391:229-38; and Aronov, et al., AnalBioanal Chem, 2008, 391:1917-30 disclose methods for detecting variousvitamin D metabolites by derivatizing the metabolites prior to massspectrometry. Methods to detect underivatized vitamin D metabolites arereported in Clarke, et al., in U.S. patent application Ser. Nos.11/101,166, filed Apr. 6, 2005, and 11/386,215, filed Mar. 21, 2006, andSingh, et al., in U.S. patent application Ser. No. 10/977,121, filedOct. 24, 2004. Reports have also been published that disclosederivatization of vitamin D₃ with Cookson-type reagents, specifically4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQ-TAD). See Aberhart, J, et al., J. Org. Chem. 1976,41(12):2098-2102, and Kamao, M, et al., J Chromatogr. B 2007,859:192-200.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of oneor more (non-metabolized) forms of vitamin D in a sample by massspectrometry, including tandem mass spectrometry. In one aspect, vitaminD from the sample is derivatized prior to analysis by mass spectrometry.In a second aspect, vitamin D from the sample is not derivatized priorto analysis by mass spectrometry.

In some embodiments of the first aspect, the methods include the stepsof: (i) subjecting Cookson-type-derivatized vitamin D in the sample toan ionization source under conditions suitable to generate one or moreprecursor ions detectable by mass spectrometry; (ii) fragmenting atleast one of said precursor ions to generate one or more fragment ionsdetectable by mass spectrometry; (iii) determining the amount of one ormore of the precursor and fragment ions by mass spectrometry; and (iv)relating the amount of ions determined in step (iii) to the amount of avitamin D in the sample. In these methods, the sample is subjected to aCookson-type derivatization reagent under conditions sufficient togenerate Cookson-type-derivatized vitamin D prior to step (i). In someembodiments, the Cookson-type-derivatized-vitamin D is subjected to anextraction column and an analytical column prior to ionization. Inrelated embodiments, the analytical column may be a high performanceliquid chromatography (HPLC) column.

In some embodiments of the first aspect, the methods include the stepsof (i) subjecting the sample to turbulent flow liquid chromatography(TFLC); (ii) subjecting Cookson-type-derivatized vitamin D from thesample an ionization source under conditions suitable to generate one ormore ions detectable by mass spectrometry; (iii) determining the amountof one or more of the Cookson-type-derivatized vitamin Dions by massspectrometry; and (iv) relating the amount of Cookson-type-derivatizedvitamin Dions determined in step (iii) to the amount of a vitamin D inthe sample. In these embodiments, the sample is subjected to aCookson-type derivatizing reagent under conditions sufficient togenerate Cookson-type-derivatized vitamin D in the sample prior to step(i). In some embodiments, the sample is subjected to high performanceliquid chromatography (HPLC) after step (i) but prior to step (ii).

In some embodiments, the Cookson-type derivatizing reagent is4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). In some of theseembodiments, the one or more precursor ions comprise one or more ionsselected from the group consisting of ions with a mass/charge ratio(m/z) of 572.4±0.5 and 560.4±0.5. In some of these embodiments, one ormore fragment ions comprise an ion with a mass/charge ratio (m/z) of298.1±0.5.

In embodiments where vitamin D comprises vitamin D₂, one or moreCookson-type-derivatized vitamin Dions may comprise a precursor ion witha mass/charge ratio (m/z) of 572.3±0.5 and a fragment ion with amass/charge ratio (m/z) of 298.1±0.5. In embodiments where vitamin Dcomprises vitamin D₃, one or more Cookson-type-derivatized vitamin Dionsmay comprise a precursor ion with a mass/charge ratio (m/z) of 560.3±0.5and a fragment ion with a mass/charge ratio (m/z) of 298.1±0.5. Inembodiments where vitamin D comprises vitamin D₂ and vitamin D₃, one ormore precursor ions may comprise a vitamin D₂ precursor ion with amass/charge ratio (m/z) of 572.4±0.5 and a vitamin D₃ precursor ion witha m/z of 560.4±0.5; and one or more fragment ions may comprise a vitaminD₂ fragment ion with a m/z of 298.1±0.5 and a vitamin D₃ fragment ionwith a m/z of 298.1±0.5.

In some embodiments of the second aspect, the invention provides methodsfor determining the amount of vitamin D in a sample by tandem massspectrometry without ionization and detection of derivatized vitamin D.In some embodiments, the vitamin D comprises vitamin D₂; these methodsinclude the steps of: subjecting vitamin D₂ from a sample to anionization source under conditions suitable to generate one or moreprecursor ions detectable by mass spectrometry selected from the groupconsisting of ions with a mass to charge ratio (m/z) of 397.2±0.5 or379.2±0.5; fragmenting at least one of said precursor ions to generateone or more fragment ions detectable by mass spectrometry; (iii)determining the amount of one or more of the ions generated in steps (i)and (ii) by mass spectrometry; and (iv) relating the presence of vitaminD₂ ions determined in step (iii) to the presence of vitamin D₂ in thesample. In these methods, if the fragmented precursor ions comprise anion with m/z of 397.2±0.5, the fragment ions comprise one or more ionsselected from the group consisting of ions with m/z of 159.0±0.5,146.9±0.5, 133.1±0.5, and 121.0±0.5. In these methods, if the fragmentedprecursor ions comprise an ion with m/z of 379.2±0.5, the fragment ionscomprise one or more ions selected from the group consisting of ionswith m/z of 283.2±0.5, 187.3±0.5, 175.2±0.5, and 159.0±0.5. In someembodiments, vitamin D₂ from the sample may be subjected to anextraction column, such as a solid phase extraction (SPE) column or aturbulent flow liquid chromatography (THLC) column, prior to ionization.In some embodiments, vitamin D₂ from the sample is further subjected toan analytical column, such as a high performance liquid chromatography(HPLC) column, prior to ionization.

In other embodiments of the second aspect, vitamin D comprises vitaminD₃; these methods include the steps of: (i) subjecting vitamin D₃ from asample to an ionization source under conditions suitable to generate oneor more precursor ions detectable by mass spectrometry selected from thegroup consisting of ions with a mass to charge ratio (m/z) of 385.2±0.5or 367.2±0.5; (ii) fragmenting at least one of the precursor ions togenerate one or more fragment ions detectable by mass spectrometry;(iii) determining the amount of one or more of the ions generated insteps (i) and (ii) by mass spectrometry; and (iv) relating the presenceof vitamin D₃ ions determined in step (iii) to the presence of vitaminD₃ in the sample. In these methods, if the fragmented precursor ionscomprise an ion with m/z of 385.2±0.5, the fragment ions comprise one ormore ions selected from the group consisting of ions with m/z of159.0±0.5, 147.0±0.5, 133.1±0.5, and 107.1±0.5. If the fragmentedprecursor ions comprise an ion with m/z of 367.2±0.5, the fragment ionscomprise one or more ions selected from the group consisting of ionswith m/z of 172.2±0.5, 145.0±0.5, and 119.1±0.5. In some embodiments,vitamin D₃ from the sample may be subjected to an extraction column,such as a solid phase extraction (SPE) column or a turbulent flow liquidchromatography (THLC) column, prior to ionization. In some embodiments,vitamin D₃ from the sample is further subjected to an analytical column,such as a high performance liquid chromatography (HPLC) column, prior toionization.

In the methods described herein, mass spectrometry may be tandem massspectrometry. In embodiments utilizing tandem mass spectrometry, tandemmass spectrometry may be conducted by any method known in the art,including for example, multiple reaction monitoring, precursor ionscanning, or product ion scanning.

In embodiments which utilize an extraction column, the extraction columnmay be a solid phase extraction (SPE) column; such as a turbulent flowliquid chromatography (TFLC) column. In some embodiments, which utilizetwo or more of an extraction column, an analytical column, and anionization source, the utilized components may be connected in anon-line fashion to allow for automated sample processing and analysis.

Cookson-type derivatizing reagents useful for certain embodiments may beselected from the group consisting of4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-methyl-1,2,4-triazoline-3,5-dione (MTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopicallylabeled variants thereof. In preferred embodiments, the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or anisotopically labeled variant thereof. In certain preferred embodiments,the Cookson-type derivatizing reagent is4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or an isotopically labeledvariant thereof.

As used herein, the term “vitamin D” refers to any one or more naturallyoccurring or synthetic analog of vitamin D which are non-metabolized.This is in contrast to vitamin D metabolites which are identified byspecific chemical modification (e.g., 25-hydroxyvitamin D and1α,25-dihydroxyvitamin D) which occurs during metabolism.Non-metabolized vitamin D may also be referred to as “nutritional”vitamin D to distinguish from metabolized forms. Reference to vitamin Dwithout specifying a metabolized form is a reference to non-metabolizedforms.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Thus, a derivatizing agent is an agent that may be reactedwith another substance to derivatize the substance. For example,4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is a derivatizing reagentthat may be reacted with vitamin D to form a PTAD-derivatized vitamin D.

As used here, the names of derivatized forms vitamin D include anindication as to the nature of derivatization. For example, the PTADderivative of vitamin D₂ is indicated as PTAD-vitamin D₂ (orPTAD-derivatized vitamin D₂).

As used herein, a “Cookson-type derivatizing agent” is a 4-substituted1,2,4-triazoline-3,5-dione compound. Exemplary Cookson-type derivatizingagents include 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-methyl-1,2,4-triazoline-3,5-dione (MTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD). Additionally,isotopically labeled variants of Cookson-type derivatizing agents may beused in some embodiments. For example, the ¹³C₆-PTAD isotopic variant is6 mass units heavier than normal PTAD and may be used in someembodiments. Derivatization of vitamin D metabolites by Cookson-typereagents can be conducted by any appropriate method. See, e.g.,Holmquist, et al., U.S. patent application Ser. No. 11/946,765, filedDec. 28, 2007; Yeung B, et al., J Chromatogr. 1993, 645(1):115-23;Higashi T, et al., Steroids. 2000, 65(5):281-94; Higashi T, et al., BiolPharm Bull. 2001, 24(7):738-43; Higashi T, et al., J Pharm Biomed Anal.2002, 29(5):947-55; Higashi T, et al., Anal. Biochanal Chem, 2008,391:229-38; and Aronov, et al., Anal Bioanal Chem, 2008, 391:1917-30.

Vitamin D may refer to one or more forms of vitamin D, such as vitaminD₂ and/or vitamin D₃. In embodiments were a sample comprises a pluralityof vitamin D forms, the plurality of vitamin D forms may be ionizedsimultaneously. For example, in some embodiments, the amounts of vitaminD₂ and vitamin D₃ are determined in the same sample. In theseembodiments, (derivatized or underivatized) vitamin D₂ and vitamin D₃may be ionized simultaneously.

The term “simultaneous” as applied to simultaneously detecting theamount of two or more analytes from a sample means acquiring datareflective of the amount of the two or more analytes in the sample fromthe same sample injection. The data for each analyte may be acquiredsequentially or in parallel, depending on the instrumental techniquesemployed. For example, a single sample containing two analytes may beinjected into a HPLC column, which may then elute each analyte one afterthe other, resulting in introduction of the analytes into a massspectrometer sequentially. Determining the amount of each of these twoanalytes is simultaneous for the purposes herein, as both analytesresult from the same sample injection into the HPLC.

Vitamin D may be found in the circulation of an animal and/or may begenerated by a biological organism, such as an animal. As such, samplesmay be obtained, for example, from a patient; that is, a living person,male or female, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. Preferred samples maybe biological samples; particularly biological fluid samples such asserum or plasma. The methods presented herein may be used to determinethe amount of vitamin D present in a sample when taken from the human.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI), laser diode thermal desorption (LDTD), or electrosprayionization (ESI), may be used in embodiments of the present invention.In certain preferred embodiments, vitamin D metabolites are measuredusing APCI or LDTD in positive ion mode.

In preferred embodiments, one or more separately detectable internalstandards are provided in the sample, the amount of which are alsodetermined in the sample. In these embodiments, all or a portion of boththe analyte(s) of interest and the internal standard(s) present in thesample are ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each are detected bymass spectrometry. Preferably, the internal standard(s) are one or moreof vitamin D₂-[6, 19, 19]-²H₃, vitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆,vitamin D₃-[6, 19, 19]-²H₃, and vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆.

One or more separately detectable internal standards may be provided inthe sample prior to treatment with a Cookson-type derivatizing reagent(if applicable), or any purification of the analyte(s) from the sample.In these embodiments, the one or more internal standards may undergoderivatization and/or purification along with the endogenous vitamin D,in which case ions of the derivatized and/or purified internal standardsare detected by mass spectrometry. In these embodiments, the presence oramount of ions generated from the analyte of interest may be related tothe presence or amount of analyte of interest in the sample. In someembodiments, the internal standards may be isotopically labeled versionsof vitamin D, such as vitamin D₂-[6, 19, 19]-²H₃, vitamin D₂-[26, 26,26, 27, 27, 27]-²H₆, vitamin D₃-[6, 19, 19]-²H₃, and vitamin D₃-[26, 26,26, 27, 27, 27]-²H₆.

Ions detectable in a mass spectrometer may be generated for each of theexemplary internal standards listed above. Exemplary spectra generatedfor several exemplary internal standards are discussed in Examples 8 and9, and shown in FIGS. 6-7, 9-10, 12-13, and 15-16.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. For example, vitamin D₂-[6, 19, 19]-²H₃ and vitaminD₃-[6, 19, 19]-²H₃ has masses of about 3 mass units higher than vitaminD₂ and vitamin D₃, respectively. The isotopic label can be incorporatedat one or more positions in the molecule and one or more kinds ofisotopic labels can be used on the same isotopically labeled molecule.

In other embodiments, the amount of vitamin Dion or ions may bedetermined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with one or more of vitamin D₂-[6, 19, 19]-²H₃, vitamin D₂-[26,26, 26, 27, 27, 27]-²H₆, vitamin D₃-[6, 19, 19]-²H₃, and vitamin D₃-[26,26, 26, 27, 27, 27]-²H₆. External standards typically will undergo thesame treatment and analysis as any other sample to be analyzed,including treatment with one or more Cookson-type reagents prior to massspectrometry in embodiments wherein derivatized vitamin D is detected.

In certain embodiments, the lower limit of quantitation (LLOQ) ofvitamin D₂ and vitamin D₃ are less than 10 ng/mL; preferably less than 5ng/mL; preferably less than 2 ng/mL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. In a preferredembodiment the analytical column contains particles of about 5 μm indiameter. Such columns are often distinguished from “extractioncolumns”, which have the general purpose of separating or extractingretained material from non-retained materials in order to obtain apurified sample for further analysis.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces;” 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to a form of ionization where the mechanism for the ionization ofmolecule M is photon absorption and electron ejection to form themolecular ion M+. Because the energy of the absorbed photon is typicallyjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser diode thermal desorption (LDTD) is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase samplemay then be drawn into an ionization source, where the gas phase sampleis ionized in preparation for analysis in the mass spectrometer. Whenusing LDTD, ionization of the gas phase sample may be accomplished byany suitable technique known in the art, such as by ionization with acorona discharge (for example by APCI).

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show exemplary chromatograms for PTAD-vitamin D₂,PTAD-vitamin D₃, and PTAD-vitamin D₃-[6, 19, 19]-²H₃ (internalstandard), respectively. Details are discussed in Example 3.

FIGS. 2A and 2B show exemplary calibration curves for vitamin D₂ andvitamin D₃ in serum samples determined by methods described in Example3.

FIG. 3 shows a plots of coefficient of variation versus concentrationfor vitamin D₂ and vitamin D₃. Details are described in Example 5.

FIG. 4 shows results of comparative studies of analysis of vitamin D₃ indifferent sample matrices. Details are described in Example 11.

FIG. 5A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for ionization of vitamin D₂. FIG. 5B shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₂ precursor ion with m/z of about397.2. FIG. 5C shows an exemplary product ion spectra (covering the m/zrange of about 100 to 400) for fragmentation of the vitamin D₂ precursorion with m/z of about 379.2. Details are described in Example 14.

FIG. 6A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for vitamin D₂-[6, 19, 19]-²H₃ ions. FIG. 6B shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₂-[6, 19, 19]-²H₃ precursor ionwith m/z of about 400.2. FIG. 6C shows an exemplary product ion spectra(covering the m/z range of about 100 to 400) for fragmentation of thevitamin D₂-[6, 19, 19]-²H₃ precursor ion with m/z of about 382.2 Detailsare described in Example 14.

FIG. 7A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for vitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ ions. FIG.7B shows an exemplary product ion spectra (covering the m/z range ofabout 100 to 400) for fragmentation of the vitamin D₂-[26, 26, 26, 27,27, 27]-²H₆ precursor ion with m/z of about 403.2. FIG. 7C shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆precursor ion with m/z of about 385.2. Details are described in Example14.

FIG. 8A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for ionization of vitamin D₃. FIG. 8B shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₃ precursor ion with m/z of about385.2. FIG. 8C shows an exemplary product ion spectra (covering the m/zrange of about 100 to 400) for fragmentation of the vitamin D₃ precursorion with m/z of about 367.2. Details are described in Example 14.

FIG. 9A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for vitamin D₃-[6, 19, 19]-²H₃ ions. FIG. 9B shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₃-[6, 19, 19]-²H₃ precursor ionwith m/z of about 388.2. FIG. 9C shows an exemplary product ion spectra(covering the m/z range of about 100 to 400) for fragmentation of thevitamin D₃-[6, 19, 19]-²H₃ precursor ion with m/z of about 370.2 Detailsare described in Example 14.

FIG. 10A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 300 to 450) for vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ ions. FIG.10B shows an exemplary product ion spectra (covering the m/z range ofabout 100 to 400) for fragmentation of the vitamin D₃-[26, 26, 26, 27,27, 27]-²H₆ precursor ion with m/z of about 391.2. FIG. 10C shows anexemplary product ion spectra (covering the m/z range of about 100 to400) for fragmentation of the vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆precursor ion with m/z of about 373.2. Details are described in Example14.

FIG. 11A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₂ ions. FIG. 11B shows an exemplaryproduct ion spectra (covering the m/z range of about 250 to 350) forfragmentation of the PTAD-vitamin D₂ precursor ion with m/z of about572.2. Details are described in Example 15.

FIG. 12A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₂-[6, 19, 19]-²H₃ ions. FIG. 12Bshows an exemplary product ion spectra (covering the m/z range of about250 to 350) for fragmentation of the PTAD-vitamin D₂-[6, 19, 19]-²H₃precursor ion with m/z of about 575.2. Details are described in Example15.

FIG. 13A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ ions.FIG. 13B shows an exemplary product ion spectra (covering the m/z rangeof about 250 to 350) for fragmentation of the PTAD-vitamin D₂-[26, 26,26, 27, 27, 27]-²H₆ precursor ion with m/z of about 578.2. Details aredescribed in Example 15.

FIG. 14A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₃ ions. FIG. 14B shows an exemplaryproduct ion spectra (covering the m/z range of about 250 to 350) forfragmentation of the PTAD-vitamin D₃ precursor ion with m/z of about560.2. Details are described in Example 15.

FIG. 15A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₃-[6, 19, 19]-²H₃ ions. FIG. 15Bshows an exemplary product ion spectra (covering the m/z range of about250 to 350) for fragmentation of the PTAD-vitamin D₃-[6, 19, 19]-²H₃precursor ion with m/z of about 563.2. Details are described in Example15.

FIG. 16A shows an exemplary Q1 scan spectrum (covering the m/z range ofabout 500 to 620) for PTAD-vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ ions.FIG. 16B shows an exemplary product ion spectra (covering the m/z rangeof about 250 to 350) for fragmentation of the PTAD-vitamin D₃-[26, 26,26, 27, 27, 27]-²H₆ precursor ion with m/z of about 566.2. Details aredescribed in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring vitamin D in a sample. Morespecifically, mass spectrometric methods are described for detecting andquantifying vitamin D in a sample. The methods may utilize Cookson-typereagents, such as PTAD, to generate derivatized vitamin D. However, insome methods, no derivatizing agent is used, and underivatized vitaminD₂ and/or vitamin D₃ are detected by mass spectrometry.

The methods may use an extraction chromatography technique, such asturbulent flow liquid chromatography (TFLC), to perform a purificationof underivatized or derivatized vitamin D₂ and/or vitamin D₃, combinedwith methods of mass spectrometry (MS), thereby providing ahigh-throughput assay system for detecting and quantifying vitamin D₂and/or vitamin D₃ in a sample. Alternatively, in some methods, nochromatography, including extraction chromatography, is necessary forsample analysis. In these methods, the underivatized or derivatizedvitamin D₂ and/or vitamin D₃ is ionized with LDTD. Preferred embodimentsare particularly well suited for application in large clinicallaboratories for automated vitamin D quantification.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma (including EDTA and heparin plasma)and serum; most preferably serum. Such samples may be obtained, forexample, from a patient; that is, a living person, male or female,presenting oneself in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition.

The present invention also contemplates kits for a vitamin Dquantitation assay. A kit for a vitamin D quantitation assay may includea kit comprising the compositions provided herein. For example, a kitmay include packaging material and measured amounts of a Cookson-typereagent and an isotopically labeled internal standard, in amountssufficient for at least one assay. Typically, the kits will also includeinstructions recorded in a tangible form (e.g., contained on paper or anelectronic medium) for using the packaged reagents for use in a vitaminD quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, vitamin D may beenriched relative to one or more other components in the sample (e.g.protein) by various methods known in the art, including for example,liquid chromatography, filtration, centrifugation, thin layerchromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving vitamin D in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, samples, such as plasma or serum, may be purifiedby a hybrid protein precipitation/liquid-liquid extraction method. Inthese embodiments, a sample is mixed with methanol, ethyl acetate, andwater, and the resulting mixture is vortexed and centrifuged. Theresulting supernatant is removed, dried to completion and reconstitutedin acetonitrile. The purified vitamin D may then be derivatized with anyCookson-type reagent, preferably PTAD or an isotopically labeled variantthereof.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with derivatized vitamin D. The chromatographiccolumn typically includes a medium (i.e., a packing material) tofacilitate separation of chemical moieties (i.e., fractionation). Themedium may include minute particles, or may include a monolithicmaterial with porous channels. A surface of the medium typicallyincludes a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bonded,cyano bonded surface, or highly pure silica surface. Alkyl bondedsurfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. Inpreferred embodiments, the column is a highly pure silica column (suchas a Thermo Hypersil Gold Aq column). The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. Thesample may be supplied to the inlet port directly, or from an extractioncolumn, such as an on-line SPE cartridge or a TFLC extraction column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with an alkyl bondedanalytical column chromatographic system. In certain preferredembodiments, a highly pure silica column (such as a Thermo Hypersil GoldAq column) is used. In certain preferred embodiments, HPLC and/or TFLCare performed using HPLC Grade water as mobile phase A and HPLC Gradeethanol as mobile phase B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, an extraction column may be used for purificationof vitamin D metabolites prior to mass spectrometry. In suchembodiments, samples may be extracted using a extraction column whichcaptures the analyte, then eluted and chromatographed on a secondextraction column or on an analytical HPLC column prior to ionization.For example, sample extraction with a TFLC extraction column may beaccomplished with a large particle size (50 μm) packed column. Sampleeluted off of this column may then be transferred to an HPLC analyticalcolumn for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

In some embodiments, protein precipitation is accomplished with a hybridprotein precipitation/liquid-liquid extraction method which includesmethanol protein precipitation and ethyl acetate/water extraction fromserum. The resulting vitamin D metabolites may be derivatized prior tobeing subjected to an extraction column. Preferably, the hybrid proteinprecipitation/liquid-liquid extraction method and the extraction columnare connected in an on-line fashion. In preferred embodiments, theextraction column is a C-8 extraction column, such as a CohesiveTechnologies C8XL online extraction column (50 μm particle size, 0.5×50mm) or equivalent. The eluent from the extraction column may then beapplied to an analytical LC column, such as a HPLC column in an on-linefashion, prior to mass spectrometric analysis. Because the stepsinvolved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, derivatized vitamin D may be ionized by anymethod known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), Laserdiode thermal desorption (LDTD), fast atom bombardment (FAB), liquidsecondary ionization (LSI), matrix assisted laser desorption ionization(MALDI), field ionization, field desorption, thermospray/plasmasprayionization, surface enhanced laser desorption ionization (SELDI),inductively coupled plasma (ICP) and particle beam ionization. Theskilled artisan will understand that the choice of ionization method maybe determined based on the analyte to be measured, type of sample, thetype of detector, the choice of positive versus negative mode, etc.

Derivatized vitamin D may be ionized in positive or negative mode. Inpreferred embodiments, derivatized vitamin D is ionized by APCI or LDTDin positive ion mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, mass transitions resulting from collisioninduced dissociation or neutral loss may be monitored, e.g., multiplereaction monitoring (MRM) or selected reaction monitoring (SRM).Preferably, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and the mass/charge ratio. The voltage andamplitude may be selected so that only ions having a particularmass/charge ratio travel the length of the quadrupole, while all otherions are deflected. Thus, quadrupole instruments may act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of vitamin D.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, in preferred embodiments one or moreisotopically labeled vitamin D (e.g., vitamin D₂-[6, 19, 19]-²H₃ andvitamin D₃-[6, 19, 19]-²H₃) may be used as internal standards. Numerousother methods for relating the amount of an ion to the amount of theoriginal molecule will be well known to those of ordinary skill in theart.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In some preferred embodiments, vitamin D in a sample is detected and/orquantified using MS/MS as follows. The samples are first purified byprotein precipitation or a hybrid protein precipitation/liquid-liquidextraction. Then, vitamin D in the purified sample is optionallyderivatized with a Cookson-type reagent, such as PTAD. The purifiedsamples are then subjected to liquid chromatography, preferably on anextraction column (such as a TFLC column) followed by an analyticalcolumn (such as a HPLC column); the flow of liquid solvent from achromatographic column enters the nebulizer interface of an MS/MSanalyzer; and the solvent/analyte mixture is converted to vapor in theheated charged tubing of the interface. The analyte(s) (e.g.,derivatized or underivatized vitamin D) contained in the solvent, areionized by applying a large voltage to the solvent/analyte mixture. Asthe analytes exit the charged tubing of the interface, thesolvent/analyte mixture nebulizes and the solvent evaporates, leavinganalyte ions. The analyte ions, e.g. precursor ions, pass through theorifice of the instrument and enter the first quadrupole. Quadrupoles 1and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e.,selection of “precursor” and “fragment” ions in Q1 and Q3, respectively)based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is thecollision cell, where ions are fragmented. The first quadrupole of themass spectrometer (Q1) selects for ions with the mass to charge ratiosof interest. Precursor ions with the correct mass/charge ratios areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. The fragment ions generatedare passed into quadrupole 3 (Q3), where derivatized or underivatizedvitamin D fragment ions are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of a derivatizedvitamin D that may be used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of vitamin D. As described above, the relative abundance of agiven ion may be converted into an absolute amount of the originalanalyte using calibration standard curves based on peaks of one or moreions of an internal molecular standard.

EXAMPLES Example 1 Hybrid Protein Precipitation/Liquid-Liquid Extractionand Cookson-Type Derivatization

The following automated hybrid protein precipitation/liquid-liquidextraction technique was conducted on patient serum samples. Gel BarrierSerum (i.e., serum collected in Serum Separator Tubes) as well as EDTAplasma and Heparin Plasma have also been established as acceptable forthis assay.

A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used toautomate the following procedure. For each sample, 50 μL of serum wasadded to a well of a 96 well plate. Then 25 μL of internal standardcocktail (containing isotopically labeled vitamin D₃-[6, 19, 19]-²H₃)was added to each well, and the plate vortexed. Then 75 μL of methanolwas added, followed by additional vortexing. 300 μL of ethyl acetate and75 μL of water was then added, followed by additional vortexing,centrifugation, and transfer of the resulting supernatant to a new96-well plate.

The transferred liquid in the second 96-well plate was dried tocompletion under a flowing nitrogen gas manifold. Derivatization wasaccomplished by adding 100 μL of a 0.1 mg/mL solution of theCookson-type derivatization agent PTAD in acetonitrile to each well. Thederivatization reaction was allowed to proceed for approximately onehour, and was quenched by adding 100 μL of water to the reactionmixture.

Example 2 Extraction of Vitamin D with Liquid Chromatography

Sample injection was performed with a Cohesive Technologies Aria TX-4TFLC system using Aria OS V 1.5.1 or newer software.

The TFLC system automatically injected an aliquot of the above preparedsamples into a Cohesive Technologies C8XL online extraction column (50μm particle size, 005×50 mm, from Cohesive Technologies, Inc.) packedwith large particles. The samples were loaded at a high flow rate tocreate turbulence inside the extraction column. This turbulence ensuredoptimized binding of derivatized vitamin D to the large particles in thecolumn and the passage of excess derivatizing reagent and debris towaste.

Following loading, the sample was eluted off to the analytical column, aThermo Hypersil Gold Aq analytical column (5 μm particle size, 50×2.1mm), with a water/ethanol elution gradient. The HPLC gradient wasapplied to the analytical column, to separate vitamin D from otheranalytes contained in the sample. Mobile phase A was water and mobilephase B was ethanol. The HPLC gradient started with a 35% organicgradient which was ramped to 99% in approximately 65 seconds.

Example 3 Detection and Quantitation of Derivatized Vitamin D by MS/MS

MS/MS was performed on the above generated samples using a Finnigan TSQQuantum Ultra MS/MS system (Thermo Electron Corporation). The followingsoftware programs, all from Thermo Electron, were used in the Examplesdescribed herein: Quantum Tune Master V 1.5 or newer, Xcalibur V 2.07 ornewer, LCQuan V 2.56 (Thermo Finnigan) or newer, and ARIA OS v1.5.1(Cohesive Technologies) or newer. Liquid solvent/analyte exiting theanalytical column flowed to the nebulizer interface of the MS/MSanalyzer. The solvent/analyte mixture was converted to vapor in thetubing of the interface. Analytes in the nebulized solvent were ionizedby APCI.

Ions passed to the first quadrupole (Q1), which selected vitamin D₂ andvitamin D₃ precursor ions with a mass-to-charge ratio of 572.3±0.5 m/zand 560.3±0.5 m/z, respectively. Ions entering quadrupole 2 (Q2)collided with argon gas to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. Mass spectrometer settings areshown in Table 1. Simultaneously, the same process using isotopedilution mass spectrometry was carried out with internal standard,vitamin D₃-[6, 19, 19]-²H₃. The mass transitions used for detection andquantitation during validation on positive polarity and at the indicatedcollision energies are shown in Table 2.

TABLE 1 Mass Spectrometer Settings for Detection of PTAD-vitamin D₂,PTAD-vitamin D₃, and vitamin D₃-[6,19,19]-²H₃ (internal standard)(Positive Polarity) Mass Spectrometric Instrument Settings DischargeCurrent 4.0 μA Vaporizer Temperature 300° C. Sheath Gas Pressure 15 IonSweep Gas Pressure 0.0 Aux Gas Pressure 5 Capillary Temperature 300° C.Skimmer Offset −10 V Collision Pressure 1.5 mTorr Collision Cell Energy15 V

TABLE 2 Mass Transitions for PTAD derivatized vitamin D₂, vitamin D₃,and vitamin D₃-[6,19,19]-²H₃ (internal standard) (Positive Polarity)Precursor Product Analyte Ion (m/z) Ions (m/z) PTAD-vitamin D₂ 572.3 ±0.5 298.1 ± 0.5 PTAD-vitamin D₃ 560.3 ± 0.5 298.1 ± 0.5 PTAD-vitamin D₃-563.3 ± 0.5 301.1 ± 0.5 [6,19,19]-²H₃

Exemplary chromatograms for PTAD-vitamin D₂, PTAD-vitamin D₃,PTAD-vitamin D₃-[6, 19, 19]-²H₃ (internal standard), are shown in FIGS.1A, 1B, and 1C, respectively.

Exemplary calibration curves for the determination of vitamin D₂ andvitamin D₃ in serum specimens are shown in FIGS. 2A and 2B,respectively.

Example 4 Linearity of Response for Detection of Derivatized Vitamin DMS/MS

Linearity was determined by diluting four pools of serum with highendogenous concentration of either vitamin D₂ or vitamin D₃ andanalyzing dilutions of 75%, 50%, and 25% in duplicate according to themethods of Examples 1-3. Specimens may be diluted 1:4 with averagerecovery of 102%, permitting a Clinically Reportable Range (CRR) of2-240 ng/mL within precision limits of 85%-115% CV. Measured values andpercent recoveries from these studies are shown in Table 3.

TABLE 3 Data Demonstrating Linearity of Response over Dilution RangePool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Nutritional Vitamin D₂ (ng/mL(percent recovery %)) Concen- tration 100%  25.0 24.5 28.1 24.4 28.126.7 (100)   (100)   (100)   (100)   (100)   (100)   75% 17.8 17.5 21.917.3 21.2 20.1  (95.3) (95.4) (103.7)  (94.5) (100.4) (100.5) 50% 12.813.0 14.0 13.8 14.8 11.8 (102.3) (106.2)   (99.4) (112.8) (105.7) (88.9) 25%  6.7  6.2  7.7  6.7  7.3  7.4 (107.9) (101.4)  (109.6)(109.6) (104.1) (110.7) Nutritional Vitamin D₃ (ng/mL (percent recovery%)) Sample concen- tration 100%  31.2 33.5 33.4 29.1 29.6 30.6 (100)  (100)   (100)   (100)   (100)   (100)   75% 22.1 23.6 25.8 22.0 24.824.1  (94.2) (93.8) (102.8) (100.8) (111.9) (104.8) 50% 15.9 16.1 16.515.7 15.9 15.0 (101.9) (96.1)  (98.5) (107.8) (107.8)  (98.0) 25%  7.6 7.9  8.5  7.3  8.0  8.0  (97.6) (94.3) (101.8) (100.4) (108.4) (105.0)

Example 5 Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) andLimit of Detection (LOD)

The lower limit of quantitation (LLOQ) is the point where measurementsbecome quantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with a precision (i.e.,coefficient of variation (CV)) of greater than 20% and an accuracy of80% to 120%. The LLOQ was determined by assaying samples with knownanalyte concentrations (2 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL,40 ng/mL, and 60 ng/mL) in quadruplicate five times according to themethods of Examples 1-3, then evaluating the reproducibility. Analysisof the collected data indicates that samples with concentrations of lessthan 2 ng/mL yielded CVs of less than 20% for both analytes. Thus, theLLOQ of each analyte was determined to be <2 ng/mL. Data generated forthe determination of LLOQ of PTAD-vitamin D₂ and PTAD-vitamin D₃ areshown in Tables 4 and 5, respectively. The graphical representations ofCV versus concentration for both analytes are shown in FIG. 3.

TABLE 4 Determination of Lower Limit of Quantitation of Vitamin D₂Vitamin D₂ concentration Assay Assay Assay Assay Assay (ng/mL) 1 2 3 4 52 2.1 1.9 2.1 1.9 2.0 Average 2.04 1.7 2.0 2.1 1.9 2.0 Std Dev 0.2 1.92.0 2.2 2.2 2.3 CV (%) 8.5 1.9 1.9 2.1 2.5 2.2 Accuracy (%) 101.8 5 4.06.2 4.3 4.7 4.0 Average 4.7 NC 4.6 4.6 4.6 4.6 Std Dev 0.6 4.6 5.6 5.14.7 4.3 CV (%) 12.8 5.7 4.1 4.6 4.6 5.3 Accuracy (%) 94.8 10 9.8 11.210.8 11.3 10.3 Average 10.3 11.3 9.6 8.6 10.0 10.9 Std Dev 0.7 10.9 10.69.9 9.6 10.1 CV (%) 6.9 10.0 9.7 9.6 10.2 10.9 Accuracy (%) 102.6 1513.6 14.7 15.6 15.0 14.5 Average 14.6 13.0 16.2 15.6 12.7 17.4 Std Dev1.2 13.6 13.4 13.6 14.1 13.4 CV (%) 8.2 14.8 14.6 16.0 14.8 14.6Accuracy (%) 97.1 20 19.4 19.1 20.3 19.9 19.1 Average 20.2 22.0 18.620.7 21.6 19.4 Std Dev 1.0 19.0 21.1 21.9 20.8 20.1 CV (%) 4.9 20.0 19.619.3 20.7 20.4 Accuracy (%) 100.8 40 42.2 41.5 39.1 42.5 41.8 Average40.3 39.2 40.6 39.8 39.0 37.8 Std Dev 1.6 40.2 40.0 41.3 40.4 37.6 CV(%) 3.9 41.1 39.6 38.9 39.6 43.6 Accuracy (%) 100.7 60 64.4 57.8 59.159.3 57.0 Average 59.8 57.4 61.4 62.3 60.8 65.2 Std Dev 2.5 58.9 59.256.8 59.0 62.0 CV (%) 4.2 58.1 61.1 61.8 59.7 55.6 Accuracy (%) 99.7

TABLE 5 Determination of Lower Limit of Quantitation of Vitamin D₃Vitamin D₃ concentration Assay Assay Assay Assay Assay (ng/mL) 1 2 3 4 52 2.1 1.6 2.2 2.0 1.8 Average 2.03 2.1 1.8 2.2 1.8 1.8 Std Dev 0.3 1.62.3 2.0 2.3 1.7 CV (%) 14.3 2.0 2.5 2.0 2.0 2.7 Accuracy (%) 101.4 5 4.75.6 4.6 5.0 4.9 Average 5.0 7.1 4.9 4.7 5.3 4.4 Std Dev 0.6 4.6 5.0 5.45.5 5.0 CV (%) 12.2 4.6 4.7 4.3 4.7 5.1 Accuracy (%) 99.7 10 9.4 10.210.1 9.9 11.0 Average 10.0 11.6 9.6 9.3 10.3 10.0 Std Dev 0.7 10.1 10.29.2 9.0 11.3 CV (%) 7.5 9.5 8.6 10.0 9.5 10.2 Accuracy (%) 99.5 15 14.514.9 14.4 15.7 15.8 Average 15.0 14.2 16.6 15.7 12.8 15.5 Std Dev 0.915.3 13.8 14.2 15.1 14.4 CV (%) 6.3 15.7 15.1 16.1 16.0 14.2 Accuracy(%) 100.0 20 19.8 18.4 18.2 22.2 17.5 Average 19.5 19.0 19.1 25.0 19.920.9 Std Dev 1.7 17.8 20.1 20.0 20.1 18.3 CV (%) 8.9 17.8 19.7 19.7 18.618.6 Accuracy (%) 97.7 40 44.3 41.7 40.6 39.1 42.0 Average 41.0 39.340.3 38.5 40.2 38.6 Std Dev 1.8 42.6 42.6 42.0 41.7 40.1 CV (%) 4.4 42.242.1 37.8 40.3 44.0 Accuracy (%) 102.5 60 65.0 57.0 60.3 60.6 55.7Average 59.5 57.6 61.5 61.1 59.3 62.6 Std Dev 2.7 55.4 55.6 56.9 60.061.6 CV (%) 4.5 58.2 62.4 61.7 59.3 58.1 Accuracy (%) 99.2

The limit of detection (LOD) is the point where a measured value islarger than the uncertainty associated with it and is definedarbitrarily as four standard deviations (SD) from the zeroconcentration. Selectivity is the ability of an analytical method todifferentiate and quantify the analyte in the presence of othercomponents in the sample. A blank was analyzed in 20 replicatesaccording to the methods of Examples 1-3 and the resulting area ratioswere statistically analyzed to determine that the LOD for both vitaminD₂ and vitamin D₃ are 0.4 ng/mL. Data collected to determine LOD foreach analyte is shown in Table 6.

TABLE 6 Determination of Limit of Detection of Vitamin D₂ and Vitamin D₃Replicate # NVD₂ (Response Ratio) NVD₃ (Response Ratio) 1 0.012 0.014 20.004 0.005 3 0.008 0.011 4 0.005 0.004 5 0.018 0.03 6 0.004 0.001 70.007 0.006 8 0.006 0.002 9 0.014 0.006 10 0.015 0.006 11 0.003 0.008 120.005 0.004 13 0.009 0.002 14 0.017 0.016 15 0.007 0.005 16 0.006 0.01217 0.003 0.001 18 0.002 0.001 19 0.011 0.001 20 0.023 0.009 Mean 0.0090.007 SD 0.0058 0.0070 CV 65.1 97.0 Mean + 4SD 0.032 0.035 LOD 0.4 ng/mL0.4 ng/mL

Example 7 Specificity of Detection

Several samples were prepared with vitamin D₂, vitamin D₃, and spikedamounts of potentially interfering species (including vitamin Dmetabolites and related compounds) and analyzed according to the methodsof Examples 1-3. The compounds tested for potential interference arelisted in Table 7. None of the tested compounds demonstratedcross-reactivity with detection of vitamin D₂ or vitamin D₃ according tothe methods of Examples 1-3.

TABLE 7 Compounds Tested for Possible Interference with Detection ofVitamin D₂ or Vitamin D₃ Compounds Tested 1,25(OH)₂D₃ 1,25(OH)₂D₂1,25(OH)₂D₃-[6,19,19′]-²H 1,25(OH)₂D₃-[26,26,26,27,27,27]-²H1,25(OH)₂D₂-[26,26,26,27,27,27]-²H 25OHD₃ 25OHD₂ 25OHD₃-IS-[6,19,19′]-²H25OHD₂-IS-[6,19,19′]-²H 25OHD₃-IS-[26,26,26,27,27,27]-²H25OHD₂-IS-[26,26,26,27,27,27]-²H vitamin D₃-[6,19,19′]-²H vitaminD₂-[6,19,19′]-²H vitamin D₃-[26,26,26,27,27,27]-²H vitaminD₂-[26,26,26,27,27,27]-²H 1-OH-D₃(Alfacalcidiol) 1-OH-D₂(Hectoral)24,25(OH)₂D₃ 25,26(OH)₂D₃ 3-epi-25OHD₃ 3-epi-1,25(OH)₂D₃Dihydrotachysterol 1,25(OH)₂D₃-26,23-lactone Paracalcitol (Zemplar)Calcipotriene (Dovonex) 7-Dehydrocholesterol

Example 8 Reproducibility of Quantitation of Vitamin D₂ and Vitamin D₃

The intra-assay variation is defined as the reproducibility of a samplewithin an assay and was determined by assaying 20 replicates of a samplefrom each of three QC pools according to the methods of Examples 1-3.Data collected from these analyses are shown in Tables 8 and 9 forvitamin D₂ and vitamin D₃, respectively. The concentrations of theanalytes in the QC pools were determined to be 6.6 ng/mL, 20.6 ng/mL,and 52.6 ng/mL for vitamin D₂, and 4.9 ng/mL, 20.5 ng/mL, and 48.6 ng/mLfor Vitamin D₃. Statistics performed on the results yieldedreproducibility for the three QC pools at 5.1%, 4.6%, and 3.9% forvitamin D₂, and 6.4%, 4.0%, and 4.5% for vitamin D₃.

TABLE 8 Intra-Assay Variation Determination for Vitamin D₂ Low QC MediumQC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Lot 120709-L Lot120709-M Lot 120709-H 1 6.7 20.3 52.5 2 6.6 21.0 54.7 3 7.0 21.1 51.5 46.2 19.5 52.1 5 6.4 20.0 52.0 6 6.2 22.4 53.3 7 7.0 20.4 48.9 8 6.7 21.554.6 9 6.7 21.5 52.4 10 6.9 20.9 52.5 11 7.0 19.2 50.2 12 6.2 20.8 52.313 7.0 19.6 57.5 14 6.6 20.6 54.7 15 6.2 19.8 53.0 16 6.2 20.6 49.2 176.2 22.9 52.5 18 6.2 20.9 51.4 19 7.0 19.6 52.2 20 6.5 20.3 55.2 Mean6.6 20.6 52.6 SD 0.3 1.0 2.0 CV % 5.1 4.6 3.9

TABLE 9 Intra-Assay Variation Determination for Vitamin D₃ Low QC MediumQC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Lot 120709-L Lot120709-M Lot 120709-H 1 4.3 20.0 48.3 2 5.1 21.8 47.2 3 4.8 21.7 45.4 44.5 21.3 49.9 5 5.0 20.5 49.8 6 4.7 21.0 48.2 7 5.8 20.4 46.3 8 4.9 21.250.8 9 4.7 22.0 49.1 10 4.7 20.1 48.9 11 4.8 19.1 47.3 12 4.7 19.9 49.113 4.9 19.5 52.9 14 5.4 20.3 52.7 15 4.7 19.6 51.3 16 4.8 19.9 46.7 174.8 21.0 47.5 18 4.7 21.1 46.9 19 5.0 19.7 45.3 20 5.1 20.4 48.8 Mean4.9 20.5 48.6 SD 0.3 0.8 2.2 CV % 6.4 4.0 4.5

The inter-assay variation is defined as the reproducibility (CV) of asample between assays. Using the three QC pools covering the reportablerange of the assay, evaluated over 5 assays according to the methods ofExamples 1-3, the inter-assay variation (CV) for the pools wasdetermined for vitamin D₂ and vitamin D₃. For Vitamin D₂, the CVs weredetermined to be 6.7%, 5.6%, and 4.0% with mean concentrations of 6.5ng/mL, 21.1 ng/mL, and 50.5 ng/mL, respectively. For Vitamin D₃, the CVswere determined to be 6.5%, 5.9%, and 4.2% with mean concentrations of4.7 ng/mL, 20.8 ng/mL, and 46.8 ng/mL, respectively. Data collected fromthese analyses are shown in Tables 10 and 11 for vitamin D₂ and vitaminD₃, respectively. All pools met with acceptable reproducibilityrequirements of ≦15% CV.

TABLE 10 Inter-Assay Variation Determination for Vitamin D₂ Low QCMedium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Assay Lot120709-L Lot 120709-M Lot 120709-H 1 6.7 20.3 52.5 6.6 21.0 54.7 7.021.1 51.5 6.2 19.5 52.1 6.4 20.0 52.0 2 7.3 23.3 49.4 6.8 22.1 51.6 6.720.6 48.6 6.7 20.6 47.2 7.0 21.1 50.4 3 6.3 22.3 49.7 6.1 23.0 52.2 6.624.0 49.5 5.6 22.1 49.6 6.1 19.1 51.7 4 6.1 20.5 47.5 7.0 21.1 50.1 6.922.2 50.2 7.3 22.1 48.3 6.6 20.2 46.8 5 5.8 21.1 52.4 6.9 19.6 49.6 6.020.8 49.1 6.4 20.6 56.1 6.3 20.1 50.8 6 6.3 21.8 50.0 7.0 22.5 49.8 6.221.6 51.2 5.9 20.1 50.3 6.9 20.1 50.9 Mean 6.5 21.1 50.5 SD 0.4 1.2 2.0CV % 6.7 5.6 4.0

TABLE 11 Inter-Assay Variation Determination for Vitamin D₃ Low QCMedium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Assay Lot120709-L Lot 120709-M Lot 120709-H 1 4.3 20.0 48.3 5.1 21.8 47.2 4.821.7 45.4 4.5 21.3 49.9 5.0 20.5 49.8 2 4.5 23.0 46.3 4.2 22.8 47.8 4.519.8 45.1 4.4 20.1 44.4 4.7 21.1 48.7 3 4.9 21.8 44.9 5.1 20.9 46.8 5.223.9 45.5 4.4 22.1 45.7 4.6 19.3 47.1 4 4.5 20.1 43.7 4.8 21.2 44.6 5.221.9 46.1 4.7 21.3 44.1 4.7 20.5 42.8 5 4.5 20.5 49.1 5.1 20.8 46.9 4.521.3 47.3 4.8 20.6 45.8 4.2 19.7 46.1 6 4.7 19.9 49.6 4.7 18.9 47.6 4.420.2 48.9 4.4 18.6 48.5 5.2 19.4 49.4 Mean 4.7 20.8 46.8 SD 0.3 1.2 2.0CV % 6.5 5.9 4.2

Example 9 Method Correlation Studies for Quantitation of Vitamin D₂

A method correlation study was performed for quantitation of vitamin D₂according to the methods of Examples 1-3 by comparing 20 split samplesanalyzed according to the tandem mass spectrometric methods describedherein with extensive off-line extraction followed by HPLC with UVdetection. Specimens were analyzed in singles for each method. Data wasanalyzed by Linear and Deming regressions. Correlation analyses aresummarized in Table 12.

TABLE 12 Correlation Analyses for Method Comparison Vitamin D₂ (n = 20)Linear Regression y = 1.26x − 0.55 R₂ = 0.96 Deming Regression y = 1.29x− 1.31

Example 10 Interference Studies

Hemolysis Interference: The effects of hemolysis in the assay describedin Examples 1-3 were evaluated by spiking hemoglobin into serum poolscontaining elevated vitamin D₂ and vitamin D₃. A fresh blood sample wascentrifuged to yield packed red blood cells. The cells werereconstituted in deionized water and frozen to achieve cell lysis. Thiscrude hemoglobin solution was then was spiked into the pools to generatelightly (100 mg/dL) and moderately (500 mg/dL) hemolyzed samples.Specimens were analyzed in duplicate according the method in Examples1-3 and results were compared to the control pool result and a percentdifference was calculated. The data shows that none of the hemoglobinspikes was >15% different than control, for either analyte). Therefore,light to moderate hemolyzed specimens are acceptable. For raw data seeTable 13 (% Difference=(Spiked−UnSpiked)/UnSpiked×100%).

TABLE 13 Hemolysis Interference Studies Vitamin D₂ Vitamin D₃ ng/mL %Diff. ng/mL % Diff. Pool 1 Control 22.1 31.2 Light Hemolysis 24.3 9.733.8 8.5 Moderate Hemolysis 23.8 7.6 34.3 10.0 Pool 2 Control 22.3 34.8Light Hemolysis 21.3 −4.8 32.6 −6.4 Moderate Hemolysis 20.7 −7.2 33.3−4.5 Pool 3 Control 27.3 34.6 Light Hemolysis 26.7 −2.2 34.7 0.2Moderate Hemolysis 24.6 −9.8 33.4 −3.5 Pool 4 Control 23.7 30.4 LightHemolysis 22.8 −3.8 31.5 3.6 Moderate Hemolysis 21.2 −10.6 29.8 −2.0Pool 5 Control 27.7 33.9 Light Hemolysis 26.7 −3.4 33.7 −0.4 ModerateHemolysis 25.6 −7.5 33.2 −1.9 Pool 6 Control 24.2 33.6 Light Hemolysis24.3 0.3 33.5 −0.4 Moderate Hemolysis 24.1 0.5 34.1 1.3

Icteria Interference: The effects of icteria in the assay described inExamples 1-3 were evaluated by spiking bilirubin into serum poolscontaining elevated vitamin D₂ and vitamin D₃. A concentrated solutionof bilirubin was spiked into the pools to generate lightly (10 mg/dL)and moderately (50 mg/dL) icteric specimens. Specimens were analyzed induplicate according to the method in Examples 1-3 and results werecompared to the non-icteric pool result and the accuracy was calculated.The data shows that both analytes are unaffected by icteria (all valueswithin acceptable accuracy range of 85-115%). Therefore, ictericspecimens are acceptable. For raw data see Table 14 (%Difference=(Spiked−UnSpiked)/UnSpiked×100%).

TABLE 14 Icteria Interference Studies Vitamin D₂ Vitamin D₃ ng/mL %Diff. ng/mL % Diff. Pool 1 Control 22.1 31.2 Light Icteria 22.2 0.3 32.64.8 Moderate Icteria 21.5 −2.6 30.7 −1.6 Pool 2 Control 22.3 34.8 LightIcteria 22.0 −1.3 33.4 −4.0 Moderate Icteria 20.5 −8.2 32.4 −6.9 Pool 3Control 27.3 34.6 Light Icteria 28.3 3.8 35.2 1.8 Moderate Icteria 27.1−0.7 35.0 1.0 Pool 4 Control 23.7 30.4 Light Icteria 22.2 −6.3 31.7 4.0Moderate Icteria 21.7 −8.7 30.7 1.0 Pool 5 Control 27.7 33.9 LightIcteria 26.7 −3.7 32.9 −2.8 Moderate Icteria 27.1 −2.0 32.4 −4.4 Pool 6Control 24.2 33.6 Light Icteria 22.8 −5.8 31.8 −5.5 Moderate Icteria22.6 −6.4 30.5 −9.4

Lipemia Interference: The effect of lipemia in the assay described inExamples 1-3 was evaluated by spiking porcine brain extract into serumpools containing elevated vitamin D₂ and vitamin D₃. Powdered lipidsample (Avanti Polar Lipids) was dissolved into each pool to generatelightly (400 mg/dL) and moderately (2000 mg/dL) lipemic specimens.Specimens were analyzed in duplicate according to the method in Examples1-3 and results were compared to the control pool result and theaccuracy was calculated. The data shows that both analytes areunaffected by lipemia (all values within acceptable accuracy range of85-115%). For raw data see Table 15 ((%Difference=(Spiked−UnSpiked)/UnSpiked×100%).

TABLE 15 Lipemia Interference (Porcine Brain Extract) Studies Vitamin D₂Vitamin D₃ ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 31.2 LightLipemia 22.0 −0.6 32.7 4.8 Moderate Lipemia 23.0 3.8 32.7 5.0 Pool 2Control 22.3 34.8 Light Lipemia 23.0 3.2 35.4 1.6 Moderate Lipemia 24.28.3 34.7 −0.2 Pool 3 Control 27.3 34.6 Light Lipemia 28.7 5.4 35.7 2.9Moderate Lipemia 28.1 3.3 36.5 5.3 Pool 4 Control 23.7 30.4 LightLipemia 25.5 7.4 33.2 9.0 Moderate Lipemia 23.2 −2.5 32.7 7.3 Pool 5Control 27.7 33.9 Light Lipemia 27.1 −2.2 34.4 1.5 Moderate Lipemia 26.7−3.4 33.9 0.1 Pool 6 Control 24.2 33.6 Light Lipemia 26.1 8.0 34.5 2.5Moderate Lipemia 24.0 −0.7 33.6 −0.2

The effects of lipemia in the assay described in Examples 1-3 were alsoevaluated by spiking Intralipid emulsion into serum pools containingelevated vitamin D₂ and vitamin D₃. To the serum pools, Intralipid (20%emulsion) was added to generate lightly (400 mg/dL) and moderately (2000mg/dL) lipemic specimens. Specimens were analyzed in duplicate accordingto the method in Examples 1-3 and results were compared to the controlpool result and the accuracy was calculated. The data shows that bothanalytes are unaffected by lipemia (all values within acceptableaccuracy range of 85-115%). For raw data see Table 16.

TABLE 16 Lipemia Interference (Intralipid) Studies Vitamin D₂ Vitamin D₃ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 31.2 Light Lipemia 22.51.6 33.3 6.8 Moderate Lipemia 19.8 −10.4 28.8 −7.6 Pool 2 Control 22.334.8 Light Lipemia 22.4 0.4 33.5 −3.6 Moderate Lipemia 19.2 −13.8 30.5−12.3 Pool 3 Control 27.3 34.6 Light Lipemia 27.4 0.6 34.5 −0.3 ModerateLipemia 24.9 −8.6 32.5 −6.1 Pool 4 Control 23.7 30.4 Light Lipemia 23.6−0.6 31.3 3.0 Moderate Lipemia 19.9 −16.0 29.8 −2.0 Pool 5 Control 27.733.9 Light Lipemia 27.4 −1.1 32.0 −5.6 Moderate Lipemia 25.8 −6.9 31.9−5.9 Pool 6 Control 24.2 33.6 Light Lipemia 24.5 1.2 33.8 0.6 ModerateLipemia 22.8 −5.9 30.0 −10.7

Based upon the two lipemia experiments using porcine brain extract andIntralipid, lipemic specimens are acceptable.

Example 11 Specimen Type Studies

Specimens were collected from 10 sources into four different Vacutainer®containers. The Vacutainers used were Red-Top (silicon-coated serumtubes), SST (Serum Separator Tubes, which result in gel-barrier serum),EDTA tubes, and Sodium Heparin tubes.

These 40 samples were analyzed for nutritional vitamin D₃ according tothe method in Examples 1-3. Comparative results are presented in FIG. 4.The data demonstrates that all four sample types are suitable foranalysis.

Example 12 Demonstration of Routine Range for Vitamin D₃

Serum specimens from 140 patients were analyzed according to the methoddescribed in Examples 1-3 to quantitate vitamin D₃. The results rangedfrom <2 ng/mL to about 63 ng/mL vitamin D₃, with 95% of the resultsfalling within the range of <2 ng/mL to about 20 ng/mL. The results ofthese analyses are presented in Table 17.

TABLE 17 Routine Range Studies for Vitamin D₃ (in descending order) VitD₃ Patient (ng/mL) 1 62.8 2 35.8 3 24.3 4 20.2 5 15.6 6 15.6 7 15.5 814.6 9 14.3 10 12.7 11 12.7 12 12.5 13 10.5 14 10.3 15 9.8 16 9.6 17 9.618 7.9 19 7.4 20 6.6 21 6.5 22 6.4 23 5.9 24 5.8 25 5.5 26 5.4 27 5.3 285.1 29 5.1 30 5.0 31 4.9 32 4.8 33 4.6 34 4.6 35 4.4 36 4.3 37 4.2 384.2 39 4.0 40 3.9 41 3.9 42 3.9 43 3.7 44 3.6 45 3.4 46 3.3 47 3.2 483.2 49 3.2 50 3.2 51 3.2 52 3.1 53 2.8 54 2.8 55 2.7 56 2.6 57 2.6 582.5 59 2.3 60 2.2 61 2.2 62 2.2 63 2.2 64 2.1 65 2.0 66 2.0 67 2.0 682.0 69 <2 70 <2 71 <2 72 <2 73 <2 74 <2 75 <2 76 <2 77 <2 78 <2 79 <2 80<2 81 <2 82 <2 83 <2 84 <2 85 <2 86 <2 87 <2 88 <2 89 <2 90 <2 91 <2 92<2 93 <2 94 <2 95 <2 96 <2 97 <2 98 <2 99 <2 100 <2 101 <2 102 <2 103 <2104 <2 105 <2 106 <2 107 <2 108 <2 109 <2 110 <2 111 <2 112 <2 113 <2114 <2 115 <2 116 <2 117 <2 118 <2 119 <2 120 <2 121 <2 122 <2 123 <2124 <2 125 <2 126 <2 127 <2 128 <2 129 <2 130 <2 131 <2 132 <2 133 <2134 <2 135 <2 136 <2 137 <2 138 <2 139 <2 140 <2

Example 13 Recovery Studies

Mix recovery studies were performed by analysis of specimens withnaturally elevated levels of 25-hydroxyvitamin D₂ or 25-hydroxyvitaminD₃, and therefore also had some endogenous circulating vitamin D₂ orvitamin D₃. Six pairs of specimens were selected for the studies. Fromeach pair of specimens (generically referred to as specimen A andspecimen B), five samples were prepared and analyzed in quadruplicateaccording to the method in Examples 1-3. The samples corresponded to100% A, 80% A −20% B, 50% A −50% B, 20% A −80% B, and 100% B. Theresults of the recovery studies are presented in Tables 18 and 19.

TABLE 18 Mixed Specimen Recovery Studies Vitamin D₂ Vitamin D₃ SpikeMeasured Expected Recovery Measured Expected Recovery Pool Level (ng/mL)(ng/mL) (%) (ng/mL) (ng/mL) (%) 1 100% A 49.0 2.1 80/20 A/B 37.8 39.3103.8 3.3 3.5 106.1 50/50 A/B 26.0 24.7 94.8 5.6 5.5 96.9 20/80 A/B 10.510.1 96.5 7.6 7.5 98.5 100% B 0.4 8.8 2 100% A 5.0 2.6 80/20 A/B 4.9 4.285.5 6.6 6.6 99.4 50/50 A/B 2.9 2.9 101.7 11.8 12.5 106.2 20/80 A/B 1.61.7 106.2 17.7 18.5 104.3 100% B 0.9 22.4 3 100% A 8.8 3.0 80/20 A/B 7.67.4 96.7 6.6 6.6 101.2 50/50 A/B 5.4 5.2 95.8 11.3 12.1 106.7 20/80 A/B3.1 3.0 99.1 16.3 17.5 107.6 100% B 1.6 21.2 *Measured values areaverages of analysis of four aliquots.

TABLE 19 Summary of Results of Mixed Specimen Recovery Studies PoolVitamin D₂ Vitamin D₃ 1 103.8% 106.1% 94.8% 96.9% 96.5% 98.5% 2 85.5%99.4% 101.7% 106.2% 106.2% 104.3% 3 96.7% 101.2% 95.8% 106.7% 99.1%107.6% Avg 97.8% 103.0% Avg 100.4%

Example 14 Exemplary Spectra from MS/MS Analysis of Vitamin D₂ andVitamin D₃

Exemplary Q1 scan spectra from the tandem mass spectrometric analysis ofvitamin D₂, vitamin D₂-[6, 19, 19]-²H₃, and vitamin D₂-[26, 26, 26, 27,27, 27]-²H₆ are shown in FIGS. 5A, 6A, and 7A, respectively. Theseanalyses were conducted by directly injecting standard solutionscontaining the analyte of interest into a Finnigan TSQ Quantum UltraMS/MS system (Thermo Electron Corporation). A liquid chromatographymobile phase was simulated by passing 800 μL/min of 80% acetonitrile,20% water with 0.1% formic acid through an HPLC column, upstream ofintroduction of the analyte. The analytes were ionized by APCI asdescribed above. The spectra were collected by scanning Q1 across a m/zrange of about 300 to 450.

Exemplary product ion scans generated from two different precursor ionsfor each of vitamin D₂, vitamin D₂-[6, 19, 19]-²H₃, and vitamin D₂-[26,26, 26, 27, 27, 27]-²H₆ are presented in FIGS. 5B-C, 6B-C, and 7B-C,respectively. The precursor ions selected in Q1 and the collisionenergies used to generate these product ion spectra are indicated inTable 20.

Exemplary MRM transitions for the quantitation of vitamin D₂ includefragmenting a precursor ion with a m/z of about 397.2 to a product ionwith a m/z of about 159.0; and fragmenting a precursor ion with a m/z ofabout 379.2 to a product ion with a m/z of about 158.9. Exemplary MRMtransitions for the quantitation of vitamin D₂-[6, 19, 19]-²H₃ includefragmenting a precursor ion with a m/z of about 400.2 to a product ionwith a m/z of about 147.0; and fragmenting a precursor ion with a m/z ofabout 382.2 to a product ion with a m/z of about 312.2. Exemplary MRMtransitions for the quantitation of vitamin D₂-[26, 26, 26, 27, 27,27]-²H₆ include fragmenting a precursor ion with a m/z of about 403.2 toa product ion with a m/z of about 159.1; and fragmenting a precursor ionwith a m/z of about 385.2 to a product ion with a m/z of about 159.0.However, as can be seen in the product ion scans in FIGS. 5B-C, 6B-C,and 7B-C, several other product ions are generated upon fragmentation ofthe precursor ions. Additional product ions may be selected from thoseindicated in FIGS. 5B-C, 6B-C, and 7B-C to replace or augment theexemplary fragment ions. For example, additional product ions generatedby fragmentation of the vitamin D₂ precursor ion with m/z of about 397.2include ions with m/z of about 146.9, 133.1, and 121.0. Exemplaryadditional product ions generated by fragmentation of the vitamin D₂precursor ion with m/z of about 379.2 include ions with m/z of about283.2, 187.3, and 175.2.

TABLE 20 Precursor Ions and Collision Cell Energies for Fragmentation ofvitamin D₂, vitamin D₂-[6,19,19]-²H₃, and vitaminD₂-[26,26,26,27,27,27]-²H₆ Precursor Collision Cell Analyte Ion (m/z)Energy (V) vitamin D₂ 397.2, 379.2 25 vitamin D₂-[6,19,19]-²H₃ 400.2,382.2 25 vitamin D₂-[26,26,26,27,27,27]-²H₆ 403.2, 385.2 25

Exemplary Q1 scan spectra from the tandem mass spectrometric analysis ofvitamin D₃, vitamin D₃-[6, 19, 19]-²H₃, and vitamin D₃-[26, 26, 26, 27,27, 27]-²H₆ are shown in FIGS. 8A, 9A, and 10A, respectively. Theseanalyses were conducted by directly injecting standard solutionscontaining the analyte of interest into a Finnigan TSQ Quantum UltraMS/MS system (Thermo Electron Corporation). A liquid chromatographymobile phase was simulated by passing 800 μL/min of 80% acetonitrile,20% water with 0.1% formic acid through an HPLC column, upstream ofintroduction of the analyte. The spectra were collected by scanning Q1across a m/z range of about 300 to 450.

Exemplary product ion scans generated from two different precursor ionsfor each of vitamin D₃, vitamin D₃-[6, 19, 19]-²H₃, and vitamin D₃-[26,26, 26, 27, 27, 27]-²H₆ are presented in FIGS. 8B-C, 9B-C, and 10B-C,respectively. The precursor ions selected in. Q1 and the collisionenergies used to generate these product ion spectra are indicated inTable 21.

Exemplary MRM transitions for the quantitation of vitamin D₃ includefragmenting a precursor ion with a m/z of about 385.2 to a product ionwith a m/z of about 147.0; and fragmenting a precursor ion with a m/z ofabout 367.2 to a product ion with a m/z of about 159.0. Exemplary MRMtransitions for the quantitation of vitamin D₃-[6, 19, 19]-²H₃ includefragmenting a precursor ion with a m/z of about 388.2 to a product ionwith a m/z of about 147.0; and fragmenting a precursor ion with a m/z ofabout 370.2 to a product ion with a m/z of about 162.0. Exemplary MRMtransitions for the quantitation of vitamin D₃-[26, 26, 26, 27, 27,27]-²H₆ include fragmenting a precursor ion with a m/z of about 391.2 toa product ion with a m/z of about 159.1; and fragmenting a precursor ionwith a m/z of about 373.2 to a product ion with a m/z of about 159.0.However, as can be seen in the product ion scans in FIGS. 8B-C, 9B-C,and 10B-C, several other product ions are generated upon fragmentationof the precursor ions. Additional product ions may be selected fromthose indicated in FIGS. 8B-C, 9B-C, and 10B-C to replace or augment theexemplary fragment ions. For example, additional product ions generatedby fragmentation of the vitamin D₃ precursor ion with m/z of about 385.2include ions with m/z of about 159.0, 133.1, and 107.1. Exemplaryadditional product ions generated by fragmentation of the vitamin D₃precursor ion with m/z of about 367.2 include ions with m/z of about172.9, 145.0, and 119.1.

TABLE 21 Precursor Ions and Collision Cell Energies for Fragmentation ofvitamin D₃, vitamin D₃-[6,19,19]-²H₃, and vitaminD₃-[26,26,26,27,27,27]-²H₆ Precursor Collision Cell Analyte Ion (m/z)Energy (V) vitamin D₃ 385.2, 367.2 25 vitamin D₃-[6,19,19]-²H₃ 388.2,370.2 25 vitamin D₃-[26,26,26,27,27,27]-²H₆ 391.2, 373.2 25

Example 15 Exemplary Spectra from MS/MS Analysis of PTAD DerivatizedVitamin D₂ and Vitamin D₃

PTAD derivatives of vitamin D₂, vitamin D₂-[6, 19, 19]-²H₃, vitaminD₂-[26, 26, 26, 27, 27, 27]-²H₆, vitamin D₃, vitamin D₃-[6, 19, 19]-²H₃,and vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ were prepared by treatingaliquots of stock solutions of each analyte with PTAD in acetonitrile.The derivatization reactions was allowed to proceed for approximatelyone hour, and were quenched by adding water to the reaction mixture. Thederivatized analytes were then analyzed according to the procedureoutlined above in Examples 2-3.

Exemplary Q1 scan spectra from the analysis of samples containingPTAD-vitamin D₂, PTAD-vitamin D₂-[6, 19, 19]-²H₃, and PTAD-vitaminD₂-[26, 26, 26, 27, 27, 27]-²H₆ are shown in FIGS. 11A, 12A, and 13A,respectively. These analyses were conducted by directly injectingstandard solutions containing the analyte of interest into a FinniganTSQ Quantum. Ultra MS/MS system (Thermo Electron Corporation). A liquidchromatography mobile phase was simulated by passing 800 μL/min of 80%acetonitrile, 20% water with 0.1% formic acid through an HPLC column,upstream of introduction of the analyte. The analytes were ionized byAPCI as described above. The spectra were collected by scanning Q1across a m/z range of about 500 to 620.

Exemplary product ion scans generated from precursor ions for each ofPTAD-vitamin D₂, PTAD-vitamin D₂-[6, 19, 19]-²H₃, and PTAD-vitaminD₂-[26, 26, 26, 27, 27, 27]-²H₆ are presented in FIGS. 11B, 12B, and13B, respectively. The precursor ions selected in Q1 and the collisionenergies used to generate these product ion spectra are indicated inTable 22.

An exemplary MRM transition for the quantitation of PTAD-vitamin D₂includes fragmenting a precursor ion with a m/z of about 572.2 to aproduct ion with a m/z of about 297.9. An exemplary MRM transition forthe quantitation of PTAD-vitamin D₂-[6, 19, 19]-²H₃ includes fragmentinga precursor ion with a m/z of about 575.2 to a product ion with a m/z ofabout 301.0. An exemplary MRM transition for the quantitation ofPTAD-vitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ includes fragmenting aprecursor ion with a m/z of about 578.2 to a product ion with a m/z ofabout 297.9. However, as can be seen in the product ion scans in FIGS.11B, 12B, and 13B, several other product ions are generated uponfragmentation of the precursor ions. Additional product ions may beselected from those indicated in FIGS. 11B, 12B, and 13B to replace oraugment the exemplary fragment ions. For example, additional productions generated by fragmentation of the PTAD-vitamin D₂ precursor ionwith m/z of about 572.2 include ions with m/z of about 280.1.

TABLE 22 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-vitamin D₂, PTAD-vitamin D₂-[6,19,19]-²H₃, and PTAD-vitaminD₂-[26,26,26,27,27,27]-²H₆ Precursor Collision Cell Analyte Ion (m/z)Energy (V) PTAD-vitamin D₂ 572.2 15 PTAD-vitamin D₂-[6,19,19]-²H₃ 575.215 PTAD-vitamin D₂-[26,26,26,27,27,27]-²H₆ 578.2 15

Exemplary Q1 scan spectra from the analysis of samples containingPTAD-vitamin D₃, PTAD-vitamin D₃-[6, 19, 19]-²H₃, and PTAD-vitaminD₃-[26, 26, 26, 27, 27, 27]-²H₆ are shown in FIGS. 14A, 15A, and 16A,respectively. These analyses were conducted by directly injectingstandard solutions containing the analyte of interest into a FinniganTSQ Quantum Ultra MS/MS system (Thermo Electron Corporation). A liquidchromatography mobile phase was simulated by passing 800 μL/min of 80%acetonitrile, 20% water with 0.1% formic acid through an HPLC column,upstream of introduction of the analyte. The spectra were collected byscanning Q1 across a m/z range of about 500 to 620.

Exemplary product ion scans generated from precursor ions for each ofPTAD-vitamin D₃, PTAD-vitamin D₃-[6, 19, 19]-²H₃, and PTAD-vitaminD₃-[26, 26, 26, 27, 27, 27]-²H₆ are presented in FIGS. 14B, 15B, and16B, respectively. The precursor ions selected in Q1 and the collisionenergies used to generate these product ion spectra are indicated inTable 23.

An exemplary MRM transition for the quantitation of PTAD-vitamin D₃includes fragmenting a precursor ion with a m/z of about 560.2 to aproduct ion with a m/z of about 298.0. An exemplary MRM transition forthe quantitation of PTAD-vitamin D₃-[6, 19, 19]-²H₃ includes fragmentinga precursor ion with a m/z of about 563.2 to a product ion with a m/z ofabout 301.0. An exemplary MRM transition for the quantitation ofPTAD-vitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ includes fragmenting aprecursor ion with a m/z of about 566.2 to a product ion with a m/z ofabout 298.0. However, as can be seen in the product ion scans in FIGS.14B, 15B, and 16B, several other product ions are generated uponfragmentation of the precursor ions. Additional product ions may beselected from those indicated in FIGS. 14B, 15B, and 16B to replace oraugment the exemplary fragment ions. For example, additional productions generated by fragmentation of the PTAD-vitamin D₃ precursor ionwith m/z of about 560.2 include ions with m/z of about 280.0.

TABLE 23 Precursor Ions and Collision Cell Energies for Fragmentation ofPTAD-vitamin D₃, PTAD-vitamin D₃-[6,19,19]-²H₃, and PTAD-vitaminD₃-[26,26,26,27,27,27]-²H₆ Precursor Collision Cell Analyte Ion (m/z)Energy (V) PTAD-vitamin D₃ 560.2 15 PTAD-vitamin D₃-[6,19,19]-²H₃ 563.215 PTAD-vitamin D₃-[26,26,26,27,27,27]-²H₆ 566.2 15

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for determining the amount of vitamin D in a sample bytandem mass spectrometry, the method comprising the steps of: (i)subjecting Cookson-type-derivatized vitamin D from the sample to anionization source to generate one or more precursor ions detectable bymass spectrometry; (ii) fragmenting at least one of said precursor ionsto generate one or more fragment ions detectable by mass spectrometry;(iii) determining the amount of one or more precursor and fragment ionsby mass spectrometry; and (iv) relating the amount of ions determined instep (iii) to the amount of a vitamin D in the sample, wherein thesample is subjected to a Cookson-type derivatization reagent underconditions sufficient to generate Cookson-type-derivatized vitamin Dprior to step (i).
 2. The method of claim 1, wherein theCookson-type-derivatized-vitamin D is subjected to an extraction columnand an analytical column prior to ionization.
 3. The method of claim 2,wherein the extraction column is a solid-phase extraction (SPE) column.4. The method of claim 2, wherein the extraction column is a turbulentflow liquid chromatography (TFLC) column.
 5. The method of claim 2,wherein the analytical column is a high performance liquidchromatography (HPLC) column.
 6. The method of claim 2, wherein theextraction and analytical columns and the ionization source of step (i)are connected in an on-line fashion.
 7. The method of claim 1, whereinsaid ionization source is an atmospheric pressure chemical ionization(APCI) source.
 8. The method of claim 1, wherein said tandem massspectrometry is conducted as multiple reaction monitoring, precursor ionscanning, or product ion scanning.
 9. The method of claim 1, wherein theCookson-type derivatizing reagent is selected from the group consistingof 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-methyl-1,2,4-triazoline-3,5-dione (MTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopicallylabeled variants thereof.
 10. The method of claim 1, wherein theCookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD) or an isotopically labeled variant thereof.
 11. The method ofclaim 1, wherein said vitamin D comprises one or more of vitamin D₂ andvitamin D₃.
 12. The method of claim 11, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); andsaid one or more precursor ions comprise one or more ions selected fromthe group consisting of ions with a mass/charge ratio (m/z) of 572.4±0.5and 560.4±0.5.
 13. The method of claim 11, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); andsaid one or more fragment ions comprise an ion with a mass/charge ratio(m/z) of 298.1±0.5.
 14. The method of claim 1, wherein said vitamin Dcomprises vitamin D₂.
 15. The method of claim 14, wherein theCookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD); said one or more precursor ions comprise a vitamin D₂ precursorion with a mass/charge ratio (m/z) of 572.4±0.5; and said one or morefragment ions comprise a vitamin D₂ fragment ion with a m/z of298.1±0.5.
 16. The method of claim 1, wherein said vitamin D comprisesvitamin D₃.
 17. The method of claim 16, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); saidone or more precursor ions comprise a vitamin D₃ precursor ion with amass/charge ratio (m/z) of 560.4±0.5; and said one or more fragment ionscomprise a vitamin D₃ fragment ion with a m/z of 298.1±0.5.
 18. Themethod of claim 1, wherein two or more vitamin D derivatives are ionizedsimultaneously.
 19. The method of claim 18, wherein said vitamin Dcomprises vitamin D₂ and vitamin D₃.
 20. The method of claim 19, whereinthe Cookson-type derivatizing reagent is4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursorions comprise a vitamin D₂ precursor ion with a mass/charge ratio (m/z)of 572.4±0.5 and a vitamin D₃ precursor ion with a m/z of 560.4±0.5; andsaid one or more fragment ions comprise a vitamin D₂ fragment ion with am/z of 298.1±0.5 and a vitamin D₃ fragment ion with a m/z of 298.1±0.5.21. The method of claim 1, wherein said sample comprises a biologicalsample.
 22. The method of claim 21, wherein said biological sample isfrom a human, and the amount of vitamin D determined in the sample isthe amount present in the sample when taken from the human.
 23. Themethod of claim 21, wherein said sample comprises plasma or serum.
 24. Amethod for determining the amount of vitamin D in a sample by massspectrometry, the method comprising the steps of: (i) subjecting thesample to turbulent flow liquid chromatography (TFLC); (ii) subjectingCookson-type-derivatized vitamin D from the sample to an ionizationsource under conditions suitable to generate one or more ions detectableby mass spectrometry; (iii) determining the amount of one or more of theCookson-type-derivatized vitamin Dions by mass spectrometry; and (iv)relating the amount of Cookson-type-derivatized vitamin Dions determinedin step (iii) to the amount of a vitamin D in the sample, wherein thesample is subjected to a Cookson-type derivatizing reagent underconditions sufficient to generate Cookson-type-derivatized vitamin D inthe sample prior to ionization.
 25. The method of claim 24, wherein thesample is subjected to high performance liquid chromatography (HPLC)after step (i) but prior to step (ii).
 26. The method of claim 25,wherein the TFLC of step (i), the HPLC, and the ionization of step (ii)are conducted in an on-line fashion.
 27. The method of claim 24, whereinthe ionization source is an atmospheric pressure chemical ionization(APCI) source.
 28. The method of claim 24, wherein said massspectrometry is tandem mass spectrometry.
 29. The method of claim 28,wherein said tandem mass spectrometry is conducted as multiple reactionmonitoring, precursor ion scanning, or product ion scanning.
 30. Themethod of claim 24, wherein the Cookson-type derivatizing reagent isselected from the group consisting of4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),4-methyl-1,2,4-triazoline-3,5-dione (MTAD),4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopicallylabeled variants thereof.
 31. The method of claim 24, wherein theCookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD) or an isotopically labeled variant thereof.
 32. The method ofclaim 24, wherein said vitamin D comprises one or more of vitamin D₂ andvitamin D₃.
 33. The method of claim 32, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); andsaid one or more precursor ions comprise one or more ions selected fromthe group consisting of ions with a mass/charge ratio (m/z) of 572.4±0.5and 560.4±0.5.
 34. The method of claim 32, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); andsaid one or more fragment ions comprise an ion with a mass/charge ratio(m/z) of 298.1±0.5.
 35. The method of claim 24, wherein said vitamin Dcomprises vitamin D₂.
 36. The method of claim 35, wherein theCookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD); said one or more precursor ions comprise a vitamin D₂ precursorion with a mass/charge ratio (m/z) of 572.4±0.5; and said one or morefragment ions comprise a vitamin D₂ fragment ion with a m/z of298.1±0.5.
 37. The method of claim 24, wherein said vitamin D comprisesvitamin D₃.
 38. The method of claim 37, wherein the Cookson-typederivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); saidone or more precursor ions comprise a vitamin D₃ precursor ion with amass/charge ratio (m/z) of 560.4±0.5; and said one or more fragment ionscomprise a vitamin D₃ fragment ion with a m/z of 298.1±0.5.
 39. Themethod of claim 24, wherein two or more vitamin D derivatives areionized simultaneously.
 40. The method of claim 39, wherein said vitaminD comprises vitamin D₂ and vitamin D₃.
 41. The method of claim 40,wherein the Cookson-type derivatizing reagent is4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursorions comprise a vitamin D₂ precursor ion with a mass/charge ratio (m/z)of 572.4±0.5 and a vitamin D₃ precursor ion with a m/z of 560.4±0.5; andsaid one or more fragment ions comprise a vitamin D₂ fragment ion with am/z of 298.1±0.5 and a vitamin D₃ fragment ion with a m/z of 298.1±0.5.42. The method of claim 24, wherein said sample comprises a biologicalsample.
 43. The method of claim 42, wherein said biological sample isfrom a human, and the amount of vitamin D determined in the sample isthe amount present in the sample when taken from the human.
 44. Themethod of claim 42, wherein said sample comprises plasma or serum.